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The nervous system of an animal can be studied by cutting it into thin layers, but this inevitably entails the destruction of cellular structures in the tissues. The analysis of complex nerve connections is then hardly possible. The method, much more elegant, is what is called the optical "cleaning" of the various tissues with the help of chemical processes that make the animal transparent. Interesting structures in the tissue can be selectively labeled and analyzed.
At the Vienna University of Technology, a cleaning method has been developed and can be applied to insects, which is a particularly difficult task. With an improved optical layer microscope (an ultramicroscope), it is now possible to image large nerve tissue samples and take high resolution images of complex neural networks labeled with fluorescent molecules. The new method was published in the journal Nature Communications.
Fluorescent molecules
"We can learn a lot about the nervous system of animals using genetic engineering to insert special molecules into nerve tissue, which can then be turned into fluorescence," says Marko Pende, a PhD student at TU Wien (Vienna). The big question is how these fluorescent molecules can be imaged without damaging the tissue.
Ultramicroscopy is a proven method. The transparent fabrics are illuminated by a laser beam, expanded by special optical elements, creating a flat light surface in two dimensions. This surface penetrates the tissue and illuminates the fluorescent molecules that are exactly in this plane. Layer by layer, the fabric can be analyzed with this light sheet, creating a three-dimensional model from the two-dimensional frames of the computer.
"We focused on the fruit fly Drosophila melanogaster because it is of particular interest for research on the nervous system. Unfortunately, it is particularly difficult to develop a method of clarification adapted to insects ", explains Marko Pende. In order for the fabric to become transparent, it must be treated with special chemicals. In insect tissues, these products have always destroyed the molecules now. "In addition, the insect tissue contains chitin, which can hardly be made transparent. Drosophila has particularly strong pigments in his eyes.
The team of TU Wien (Vienna), as well as the University of Vienna and the University of Medicine, have managed to find a way to Drosophila Steals completely transparent without destroying fluorescent marker molecules. This was achieved with the help of improved chemical blends. "This is an important step forward for the Drosophila the research community, "says Professor Thomas Hummel from the Department of Neurobiology (University of Vienna).
The images were made possible by the pioneering optical research of Saiedeh Saghafi (TU Wien). It was able to significantly improve the ultramicroscope: The sheet of light, with which the plane is illuminated layer by layer, was about 10 microns thick. The improved ultramicroscope now produces uniform bright plates of only 3 μm in thickness over a large area. In addition, the microscope was equipped with an additional lens that modified the focal point, in the manner of a pair of glasses: "Until now, we could only focus on the outer area of the fabric, we can now take one centimeter deep Look in the tissues and always get crisp images, "says Professor Hans Ulrich Dodt, Head of the Department of Bioelectronics (TU Wien)." This will yield impressive high-resolution images that will give us important information about the Drosophila nervous system works. "
The behavior of the connectome and the fruit fly
The new technique should now help to study the so-called "connectome" of Drosophila. The connectome is the arrangement of the interconnections throughout the nervous system, the "electrical scheme" of the animal. This circuit diagram can then be associated with behavior patterns of Drosophila.
In addition, Drosophila is ideal for analyzing genes that lead to neurodegenerative diseases in humans, such as Alzheimer's disease and Parkinson's disease. Transparent flies now offer a unique opportunity to understand complex changes in various regions of the nervous system during neurodegeneration.
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Material provided by Vienna University of Technology. Note: Content can be changed for style and length.
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